Project Summary/Abstract Medical treatment of corneal disease depends upon effective penetration of topical agents into the cornea. In many instances the corneal epithelium serves as a barrier to diffusion, while in cases with epithelial defects drug penetration through the stroma can be unreliable or inadequate. This study proposes to evaluate ultrasound technology to enhance the permeability of the cornea and promote corneal uptake of selected topical medications ? riboflavin, vancomycin, and voriconazole. Riboflavin is used in corneal cross-linking, a treatment for diseases of corneal weakening that stiffens the tissue through the interaction of riboflavin with ultraviolet energy. Since the epithelium is generally removed to achieve adequate riboflavin penetration in the cornea, subjecting patients to pain and the risk of infection, there is a need for an innovation that can increase stromal riboflavin uptake while avoiding epithelial removal. Since riboflavin is autofluorescent, it can be quantified in the cornea using confocal microscopy. Vancomycin-BODIPY is a labeled fluorescent version of the highly clinically relevant antibiotic and can also be detected in the cornea. It is a larger molecule and will therefore provide additional information in optimizing ultrasound parameters for generalizability to other drugs. A distinct but pressing clinical need relates to the treatment of fungal corneal ulcers, which frequently require excision of infected tissue, due in part to poor delivery of antifungal agents, such as voriconazole, into the deep stroma. We have shown a significant increase in penetration of riboflavin into the cornea following ultrasound exposure without epithelial debridement, and in preliminary experiments we observed an impressive penetration of vancomycin-BODIPY into the stroma without epithelium, as well as an improved riboflavin penetration at lower ultrasound settings by optimizing the viscosity of the solution formulation and generating microbubbles. The goal of this study therefore is to identify clinically useful ultrasound treatment regimes for riboflavin, vancomycin, and voriconazole delivery to improve patient care in a range of corneal conditions. In Aim 1, we will optimize ultrasound treatment parameters including frequency, intensity, duty cycle, and treatment time using an in vitro model of riboflavin and vancomycin-BODIPY application to the rabbit cornea, measuring drug permeation with confocal microscopy. In Aim 2, we will quantify the corneal delivery in vivo using the most effective settings identified in Aim 1. In Aim 3, we will test the ability of ultrasound to improve the delivery of voriconazole to the corneal stroma in a live rabbit model of Fusarium keratitis by quantifying the residual organisms in the cornea after treatment. In Aim 4 the safety of a clinically effective treatment regimen will be evaluated through clinical and histologic examination of the cornea at several time points with particular attention to endothelial cell damage or loss. The results can serve as the basis for clinical trials in patients undergoing cross-linking or with recalcitrant corneal infections and will provide proof of concept to investigate this technology for other applications, such as delivery of novel therapies for endothelial cell dysfunction.